Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids

The bioengineerined and whole matured human brain organoids stand as highly valuable three-dimensional in vitro brain-mimetic models to recapitulate in vivo brain development, neurodevelopmental and neurodegenerative diseases. Various instructive signals affecting multiple biological processes including morphogenesis, developmental stages, cell fate transitions, cell migration, stem cell function and immune responses have been employed for generation of physiologically functional cerebral organoids. However, the current approaches for maturation require improvement for highly harvestable and functional cerebral organoids with reduced batch-to-batch variabilities. Here, we demonstrate two different engineering approaches, the rotating cell culture system (RCCS) microgravity bioreactor and a newly designed microfluidic platform (µ-platform) to improve harvestability, reproducibility and the survival of high-quality cerebral organoids and compare with those of traditional spinner and shaker systems. RCCS and µ-platform organoids have reached ideal sizes, approximately 95% harvestability, prolonged culture time with Ki-67 + /CD31 + /β-catenin+ proliferative, adhesive and endothelial-like cells and exhibited enriched cellular diversity (abundant neural/glial/ endothelial cell population), structural brain morphogenesis, further functional neuronal identities (glutamate secreting glutamatergic, GABAergic and hippocampal neurons) and synaptogenesis (presynaptic-postsynaptic interaction) during whole human brain development. Both organoids expressed CD11b + /IBA1 + microglia and MBP + /OLIG2 + oligodendrocytes at high levels as of day 60. RCCS and µ-platform organoids showing high levels of physiological fidelity a high level of physiological fidelity can serve as functional preclinical models to test new therapeutic regimens for neurological diseases and benefit from multiplexing.

1. Cell composition variability is known to be a limitation of some human brain organoid protocols. In this study, the authors refer to reproducibility in terms of organoid size, which does not infer much about the quality of the differentiation and cell composition. Therefore, conclusions about reproducibility should take other parameters in consideration, such as cellular composition and cellular organization, instead of just organoid size.
2. Overall presentation and description of data should be significantly improved. There is insufficient characterization that limits interpretation and conclusions. Moreover, proportion of cell types and protein expression patterns present in the study are inconsistent with what is known in the field. This data should be revised and validated more carefully. Below are some examples that require careful revision: a) Brightfield images (Fig. 2) and immunostainings throughout the manuscript raise concerns about the organoid differentiation quality. It is very hard to assess the quality of differentiation by these images and it is not very clear what information the authors want to convey with the H&E staining ( Fig. 2). Additionally, the transparent regions observed in organoids from most conditions resemble cystic formation associated with off-target differentiation. Therefore, the authors should demonstrate that the organoids are indeed cortical organoids. This can be shown by high-quality images demonstrating a high proportion of FOXG1+ cells. FOXG1 is a forebrain marker that is expressed early during cortical development (PMID: 18983967, PMID: 24277810, PMID: 23995685). Moreover, the authors should include high-quality images including other standard markers to confirm successful cortical differentiation (e.g. EMX1, PAX6, SOX2, TBR1, CTIP2, SATB2), as well as verify the presence of rosette and neural tube-like structures in multiple timepoints.
b) The authors claim that both astrocytes (GFAP+) and neurons (NEUN+) are present since day 60 of organoid development. Previous studies with human brain organoids have demonstrated that differentiation in these in vitro 3D models recapitulate endogenous development where gliogenesis happens after neurogenesis. In one of these papers, astroglia are not seen before 6 months of development in vitro (PMID: 28445462). It is possible that modifications done by the authors to the protocol change this pattern, but it is recommended that higher magnification images, including more markers per cell type, are included to better illustrate the presence of these cells in their models.
c) The authors claim presence of cell types such as microglia and oligodendrocytes that are usually not present in most protocols. It is also recommended to include higher magnification images of these cells with more markers (microglia: IBA1, P2RY12, TMEM119; oligodendrocytes: MBP, OLIG2, PLP) in order to validate their presence in the organoids. d) Staining for the postsynaptic marker, PSD95, seems unspecific in the representative images and would require co-staining with a presynaptic marker and imagining using a confocal microscope. e) KI67 staining should be nuclear; however in Fig. 6a the staining seems to be outside of the nucleus. Moreover, specifically in this panel, representative organoids depicted in the RCCS and uplatform raise concern about the quality of differentiation, as broad morphology seems cystic and irregular.
f) Font size in Fig. 1 should be increased in order to be seen. Fig. 3: i. typos (GFAB, migrolglia); ii. qRT-PCR heatmap: it is not clear how the different markers vary along the time. The way it is presented looks like that some markers that are expected later in development (S100B) are rather expressed early, and downregulated later on; iii. Specific cell marker (fold change plot): it is unclear what data is being plotted in this graph and what is the final conclusion related to this data.

g) Other examples within
3. There are multiple overstatements and generalizations throughout the text that need to be revised by the authors. See some examples below: a) "Conventional cerebral organoid maturation approaches using spinners10 and shakers11, promote self-neuronal organization and formation of cerebral ventricular spaces by providing agitation. However, these methods have major limitations, including altered nutrient diffusion and apoptotic zones due to lack of vascularization12, immune cell deficiency, matrigel dependency, low reproducibility, scalability and high variability of the induced brain components and cells13." This statement should be rephrased as some of the limitations mentioned (e.g. apoptotic zones, lack of reproducibility) have already been addressed and overcome in prior studies (including the ones cited in this sentence).
b) The authors should be particularly careful about claiming "molecular and functional maturation of cerebral brain organoids". Not enough data is provided to make these claims and some of the available data is not of sufficient quality. For instance, colocalization of the postsynaptic marker, PSD95, and a presynaptic marker, demonstrated by confocal microscopy, would suggest that there are functional synapses present in the organoids, but would still be insufficient for concluding functional maturation of this organoids. c) In the Discussion section there is a series of overstatements: i. "(1) enriched cellular diversity (abundant neural/glial/ endothelial cell population)" > deeper characterization with higher quality images and more markers should be done to conclude this; ii. "(2) structural brain morphogenesis (complex cortical layers, radia-glial organization, preplate splitting)" > no clear demonstration of brain morphogenesis; indeed, the lack of complex structure in brain organoids is one of the limitations of the field. iii. "further functional neuronal identities (glutamatergic and hippocampal neurons) and synaptogenesis (postsynaptic interaction) during whole human brain development" > further analysis using more sophisticated technologies would be necessary to validate this claim. iv. "GFAP+/S100B+ astrocytes and NEUN+/MAP2+ mature neurons were abundant on day 60 in RCCS organoids richest in cell diversity, both RCCS and µ-platform organoids expressed CD11b+/IBA1+ microglia and MBP+/OLIG2+ oligodendrocytes at high levels as of day 60." > further characterization with more markers and higher quality images are necessary to support this claim. v. "RCCS organoids with enormous cell architectures, exhibited excellent cortical plate and SVZ architecture at day 120, where TUJ+ cells located at the basal side of the ventricle-like structures with TBR1+ cells located in the deep layer of cortical-like plates as well as well-defined progenitor zone organization, neural identity and further neuronal differentiation with NESTIN+/TBR2+/PAX6+ and SOX1+/SOX2+ cells." > it is not clear what the authors are referring to. vi. "On the other hand, N-CAD+ apical epithelial and TTR+ choroid plexus epithelial cells were populated in RCCS organoids as of day 60, while both FOXG1+ forebrain, KROX20+ hindbrain and PROX1+ hippocampus related to midbrain and hindbrain identities were observed in µ-platform organoids at day 120." > further characterization with more markers and higher quality images are necessary to support this claim.
Minor concerns: 1. The authors raise a valid point that hemodynamic forces are underexplored in the brain organoid field, despite the known biological relevance of mechanosensitive signals associated with cerebrospinal fluid flow during development. However, the authors do not explore this aspect in their data or discussion. It would be interesting to tie their data back to what is known about this in the neurodevelopmental field.
2. The authors hypothesize that microgravity and gravity-driven laminar flows enhance maturation of cerebral organoids recapitulating features of human embryonic cortical development. It would be interesting to discuss the findings that support this hypothesis.
3. "In terms of tissue structure, while the most dispersed organoid structure was observed in static culture and the most uniform distribution was observed in the shaker, the well-defined multipatterned organoid morphologies with self-organization and desired cellular architectures in the innerouter regions6,13,28 were observed in the RCCS microgravity bioreactor, followed by µ-platform and spinner systems (Fig. 2b,c,d,e,f)." It is not clear which parameters the authors used to say the latter. How was this quantified? 4. What does "neurons at different polarized states" mean? 5. Authors mentioned the presence of "cortical plate" in their organoids, but this is not clear from the images provided.
6. "Paired box protein 6 (PAX6) is a transcription factor expressed in neural stem cells and ventral forebrain radial glial progenitor cells. Although expressed in all brain regions, the highest expression level is reported at the cerebellum, which is involved in glial cell differentiation55." This sentence is confusing: it is unclear the relationship between glial cell differentiation and the cerebellum.
7. "CTIP2, SATB2, and TBR1 were upregulated at protein and RNA levels which indicated cortical neuronal differentiation in both RCCS and µ-platform organoids mainly on day 120 (Fig. 4, Supp. Fig.  3), supporting the spatial distribution and functional organization of cortical neurons61." It has been demonstrated in 3D brain organoid cultures that generation of the different cortical neuron subtypes is not necessarily accompanied by structural/functional organization in the stereotypical layers of the human cerebral cortex (PMID: 24277810). Therefore, this sentence should be revised.
8. In the methods, the authors mentioned that some modifications were done to the organoid protocol, but they do not mention what was done.
Reviewer #2 (Remarks to the Author): Saglam-Metiner et al. reported that RCCS microgravity bioreactor and μ-platform presented improvement of the harvestability, scalability, reproducibility, and survival of cerebral organoids, as well as functional maturation. It is assumed that comparing these various 3D culture methods with each other required a lot of effort, and the results should provide important information to the scientific research field. However, their findings are just observations, and quantitative data are lacking. Statistical analysis of quantitative data is a basic requirement for scientific consideration. Furthermore, there are no data to allow the evaluation of physiological function, although the authors mentioned that their organoids have achieved functional maturity. There remain many issues to be resolved before reaching the conclusion the authors proposed.
Major points: 1. The authors should add the quantitative data to explain the improvement of harvestability, scalability, reproducibility, and survival of cerebral organoids to compare the respective culture conditions, and they should present the results of statistical analysis. 2. Although the authors described organoid-maturation, they only presented immunostaining and western blot data to show protein expression related to synapses such as PSD95. The authors should present the data of physiological analysis to show that their organoids have acquired functional maturation as neurons. 3. Although the authors mentioned that a layered structure of brain has formed, the figures showed no such structure but only a scattering of cells expressing markers for the cortical layers. The authors should correct the description. 4. The authors should provide the details of the equipment, and especially the μ-platform that was reported as being newly constructed in this paper. 5. The authors should compare the cell-type characteristics and maturation status in single-cell resolution methods like single-cell RNA sequence or detailed IFC investigation because organoid contains various types of brain cells. Quantification of these data will be helpful for understanding the beneficial points of the presented platform. 6. The authors attempted to characterize different culture platforms by hydrodynamic analysis. This analysis clarifies the characteristics of how the medium moves and can be considered to be useful data. However, what this analysis reveals is limited to phenomena outside of organoids, such as the distribution of shear stress. This analysis unfortunately does not help us to understand what physical effects occur inside individual organoids and, as a result, affect differentiation and maturation. Care should be taken so as not to overstate this point, and to improve the description to avoid misunderstandings.
Minor points 1. Signal/background ratios of images are not enough. Revise the setting when acquiring images. 2. The shape and marginal border of organoids are irregular, and these organoids appeared to be dying in Figures 3-6. The authors can investigate and describe what happens, e.g. cellular migration, cell death, unique differentiation, etc. 3. In Figure 5b, PROX1 appears to highlights the thalamus and cerebellum. Is that correct? 1

Journal: Communications Biology
Manuscript #: COMMSBIO-22-1668-T Title of Paper: Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids Thank you for giving the opportunity to submit our revised manuscript titled "Spatio-temporal dynamics enhance cellular diversity, neuronal function and further maturation of human cerebral organoids'' to Communications Biology. We appreciate the editor and reviewers for their valuable time and effort in our manuscript. We have revised our paper based on the insightful comments of the reviewers and highlighted the changes in the manuscript. We hope that the revised manuscript can meet the journal's publication requirements.

Reviewer #1 (Remarks to the Author):
In this manuscript, Saglam-Metiner et al. describe two novel engineering approaches (RCCS and uplatform) with the goal to improve harvestability, scalability, reproducibility and survival of human brain organoids during long term cultures. The authors start by elegantly demonstrating that the proposed models (RCCS and u-platform) exhibit less shear stress when compared to traditional cultures on shakers or spinner flasks. Moreover, the shear stress distribution in RCCS and u-platform is a lot more homogeneous than when compared to the current available systems (shakers and spinners). These novel methods offer a great potential to further improve quality and durability of human brain organoids in vitro, which have been negatively impacted by the limitations associated with current culture conditions. Despite the great potential, the manuscript needs significant improvement on the characterization of differentiation and maturation of cells within the human brain organoids to conclusively assess the value of these approaches to advance the field.
Thanks for the insightful comments, which helped to increase the quality of the manuscript. Please find our point-by-point responses to each of the comments raised.
Major concerns: 1. Cell composition variability is known to be a limitation of some human brain organoid protocols. In this study, the authors refer to reproducibility in terms of organoid size, which does not infer much about the quality of the differentiation and cell composition. Therefore, conclusions about reproducibility should take other parameters in consideration, such as cellular composition and cellular organization, instead of just organoid size We appreciate the reviewer's suggestion and agree that there should be additional data to strengthen the conclusions of the study. Therefore, we performed fluorescence-activated cell sorting (FACS) analysis of microglia and astrocytes from the RCCS organoids to support cellular composition in a revised text as Fig. 3d. Method details is given as follows; Initially, for single-cell suspension from RCCS organoids, three organoids were freshly harvested from vessels, collected to tube, washed with 1x PBS and immersed in 1 mL DMEM/F12 included papain (18.6 U/mL,P3125, and DNAse 1 (337 U/mL, EN0521, Thermo Fisher) at 37°C for 30 min on shaker. It was mechanically pipetted and vortexed at ten-minute intervals under sterile conditions. Next, 2% FBS was added to stop the enzymatic reaction and the resulting singlecell suspension was centrifuged at 400 rfc for 5 min. An additional incubation was made in PBS buffer (included 2 mM EDTA, 1% FBS and 337 U/mL DNAse 1, pH 7.4) at RT for 15 min, and singlecells were passed through a 70 µm cell-strainer. Then, single-cells were labeled with CD45 and CD11b primary antibodies for microglia sorting and were labeled with GFAP primary antibody for astrocyte cell sorting (Supp. Additionally, we added a graph to highlight the harvestability results in Fig. 2b and detailed all of the results in the text under the heading "Laminar flow increases reliable production of high-quality organoids with the highest harvestability, reproducibility and reduced batch-to-batch variabilities" as follows; On the 15th day of culture, generated organoids (derived from two different passage numbers of iPSCs at two different times) were transferred to RCCS, µ-platform, spinner and shaker, along with static culture as the control group (considered as day 0 of maturation), in order to determine the best dynamic system for physical and functional maturation of cerebral organoids, which were cultured for 120 days. Organoid size distribution variability is known to be a limitation of traditional human brain organoid protocols, as well as cellular composition and organization. Therefore, organoid reproducibility and batch-to-batch variability was firstly examined in terms of harvestability, macroscopic-microscopic observations and organoid size distribution (Fig. 2b,c,d,e,f,g). As such, cellular composition and cellular organization were assessed with cell sorting, immunostaining, western blot, qRT-PCR, TUNEL and glutamate secretion analyses for organoids sampled on various days (30, 60 and 120th days).
2. Overall presentation and description of data should be significantly improved. There is insufficient characterization that limits interpretation and conclusions. Moreover, proportion of cell types and protein expression patterns present in the study are inconsistent with what is known in the field. This data should be revised and validated more carefully. Below are some examples that require careful revision: a) Brightfield images (Fig. 2) and immunostainings throughout the manuscript raise concerns about the organoid differentiation quality. It is very hard to assess the quality of differentiation by these images and it is not very clear what information the authors want to convey with the H&E staining (Fig. 2). Additionally, the transparent regions observed in organoids from most conditions resemble cystic formation associated with off-target differentiation. Therefore, the authors should demonstrate that the organoids are indeed cortical organoids. This can be shown by high-quality images demonstrating a high proportion of FOXG1+ cells. FOXG1 is a forebrain marker that is expressed early during cortical development (PMID: 18983967, PMID: 24277810, PMID: 23995685). Moreover, the authors should include high-quality images including other standard markers to confirm successful cortical differentiation (e.g. EMX1, PAX6, SOX2, TBR1, CTIP2, SATB2), as well as verify the presence of rosette and neural tubelike structures in multiple timepoints The quality of all figures has been increased and revised in the illustration program. More clear information about H&E staining (images with small magnification have been removed to avoid misunderstanding) and transparent regions added to text with white arrows to indicate organized cell nucleus and neural rosette-like structures in Fig. 2c,d,e,f,g. As well as N-CAD/FOXG1 image replacement for RCCS organoids in Fig.4a, a new staining was carried out and high-quality images demonstrating high proportion of FOXG1+ and PAX6+ cells were added to assess the quality of differentiation in Fig.6a. Also, white arrows added to indicate TBR1+/PAX6+/SOX2+/FOXG1+ and DAPI+ neural rosette and neural tube-like structures in Fig b) The authors claim that both astrocytes (GFAP+) and neurons (NEUN+) are present since day 60 of organoid development. Previous studies with human brain organoids have demonstrated that differentiation in these in vitro 3D models recapitulate endogenous development where gliogenesis happens after neurogenesis. In one of these papers, astroglia are not seen before 6 months of development in vitro (PMID: 28445462). It is possible that modifications done by the authors to the protocol change this pattern, but it is recommended that higher magnification images, including more markers per cell type, are included to better illustrate the presence of these cells in their models.
Thanks for pointing this out. The quality of all figures has been increased and revised in the illustration program. The GFAP+/S100B+ astrocytes and NEUN+/MAP2+ mature neurons were examined in detail by immunofluorescence, western blot or qRT-PCR analysis. Additionally, we performed FACS analysis of GFAP+ astrocytes from the RCCS organoids to support the presence of more cellular composition and added the results to revised text as Fig. 3d with 1

.3%±0.4 GFAP+ cell sorting rate.
c) The authors claim presence of cell types such as microglia and oligodendrocytes that are usually not present in most protocols. It is also recommended to include higher magnification images of these cells with more markers (microglia: IBA1, P2RY12, TMEM119; oligodendrocytes: MBP, OLIG2, PLP) in order to validate their presence in the organoids.

We added the following paragraph to the text;
To further interrogate the presence of microglial cells, the ratio of CD45/CD11b double positive cells in RCCS organoids was found to be 1.3%±0.4 with FACS analysis, indicating the positive effect of microgravity on mesodermal-microglial cell differentiation (Fig. 3d) d) Staining for the postsynaptic marker, PSD95, seems unspecific in the representative images and would require co-staining with a presynaptic marker (GABA-A) and imagining using a confocal microscope.
Thanks for the suggestion. Besides, postsynaptic PSD95 and presynaptic vGLUT1 staining, we additionally performed immunofluorescence staining with a presynaptic marker GABA-A to indicate GABAergic interneurons in organoids, and obtained high-quality images under the confocal microscope. GABA-A immunofluorescence staining is given in the results section in Fig. 5a. and details about GABA-A is provided in the results section as follows. Moreover, western blot analysis was performed to support results in Fig. 5c.

During early corticogenesis, bursts of action potentials cause spreading of giant waves of calcium influxes through the developing cortex, which is described as giant depolarizing potentials (GDPs), depending both on excitatory glutamate and gamma-aminobutyric acid (GABA) inputs.
Glutamate-dependent GDP-like events were reported in neuronal organoids. Reduced GDPs in >40 day organoids with GABA polarity switch were regarded as indicators of progressive neuronal network maturation 78 . On the other hand, the glutamate level that secreted from microglia was measured around 20 µM in healthy in vitro neuronal cultures 79,80 . Given that, the change in the uptake and consequently release rate of extracellular glutamate in 60 day RCCS and µ-platform organoids and growth in organoid sizes might be associated with more mature states. One of the distinguishing features of neuronal circuit and maturation is the switch of excitatory to inhibitory GABAergic neurotransmission. GABA-A, a presynaptic GABAergic interneuron receptor that modulates neurotransmitter release in both peripheral and central synapses 7,78,[81][82][83] , was more expressed in both RCCS and µ-platform organoids with CTIP2+ deep-layer of cortical neurons as of day 60 ( Fig. 5a,b,c). Thus, the neuronal network complexity was validated in RCCS and µ-platform organoids with the presence of both presynaptic glutamatergic VGLUT1+ and GABAergic GABA-A+ neurons which are critically involved in neuronal network oscillations, as well as postsynaptic PSD95+ neurons, GFAP+/S100B+ astrocytes and MBP+/OLIG2+ oligodendrocytes. e) KI67 staining should be nuclear; however in Fig. 6a the staining seems to be outside of the nucleus. Moreover, specifically in this panel, representative organoids depicted in the RCCS and u-platform raise concern about the quality of differentiation, as broad morphology seems cystic and irregular.
Thanks for pointing this out, we agree that the Ki-67 staining should be nuclear. Therefore, we repeated Ki-67 immunofluorescence staining for all groups and obtained images that resulted in more nuclear staining than the previous stainings. We revised Ki-67 images in Fig. 6a and the quality of all figures has been increased and revised in the illustration program. Additionally, to support the immunofluorescence staining results, western blot and qRT-PCR analysis were performed for the Ki-67 marker, which can be found in Fig. 6b,c and added to the text.
f) Font size in Fig. 1 should be increased in order to be seen. Fig.1 was increased in order to be seen g) Other examples within Fig. 3 i. typos (GFAB, migrolglia)

Thanks, we corrected all typos throughout the text
ii. qRT-PCR heatmap: it is not clear how the different markers vary along the time. The way it is presented looks like that some markers that are expected later in development (S100B) are rather expressed early, and downregulated later on qRT-PCR heatmap illustrations are revised as a system-based grouping to explicit their changes over time. The heatmaps were also colored with a monochromatic gradient that increases in intensity from low to high expression to illustrate the expression changes more clearly.
iii. Specific cell marker (fold change plot): it is unclear what data is being plotted in this graph and what is the final conclusion related to this data.
Thanks, mentioned graph is removed to avoid misunderstanding.
3. There are multiple overstatements and generalizations throughout the text that need to be revised by the authors. See some examples below: a) "Conventional cerebral organoid maturation approaches using spinners10 and shakers11, promote selfneuronal organization and formation of cerebral ventricular spaces by providing agitation. However, these methods have major limitations, including altered nutrient diffusion and apoptotic zones due to lack of vascularization12, immune cell deficiency, matrigel dependency, low reproducibility, scalability and high variability of the induced brain components and cells13." This statement should be rephrased as some of the limitations mentioned (e.g. apoptotic zones, lack of reproducibility) have already been addressed and overcome in prior studies (including the ones cited in this sentence).

Thank you for suggestion, the sentence is rephrased as follows;
However, these methods have major limitations, including altered nutrient diffusion and apoptotic zones due to lack of vascularization, immune cell deficiency, matrigel dependency, low reproducibility, scalability and high variability of the induced brain components and cells 12 . Different strategies have been developed to prevent these limitations such as by co-culturing with human umbilical vascular endothelial cells (HUVECs) 13 and human iPSC-derived endothelial cells not only to enhance oxygen/nutrient diffusion but also to leverage neural differentiation, migration and circuit formation during development 14 .
b) The authors should be particularly careful about claiming "molecular and functional maturation of cerebral brain organoids". Not enough data is provided to make these claims and some of the available data is not of sufficient quality. For instance, colocalization of the postsynaptic marker, PSD95, and a presynaptic marker, demonstrated by confocal microscopy, would suggest that there are functional synapses present in the organoids, but would still be insufficient for concluding functional maturation of this organoids We appreciate the reviewer's insightful suggestion and agree with this. Thus, we performed additional glutamate analysis to examine molecular and functional maturation of RCCS and platform organoids as depicted in Fig. 5d and details are given in the methods and results sections as follows..

6
To ascertain advanced maturation of RCCS and µ-platform organoids, we examined the release of glutamate. For this, samples of the medium (one week of used) were collected on day 60 of maturation process and analyzed with a colorimetric glutamate assay kit (MAK330-1K, Sigma-Aldrich), according to the manufacturer's instructions. Samples of fresh cerebral organoid differentiation medium were also run as negative controls.
VGLUTs are responsible for the vesicular accumulation of glutamate, the major excitatory neurotransmitter that plays critical roles in neuronal signaling and cortical development, which is uploaded into synaptic vesicles within presynaptic terminals before undergoing regulated release at the synaptic cleft 76,77 . Therefore, we decided to examine the cumulative glutamate levels in one week of used maturation medium of RCCS and µ-platform organoids at days 30 and 60 to evaluate further maturation. Significant time-dependent increases were noted in glutamate levels for both groups (p<0.0001 and p<0.01, respectively), even more in RCCS organoids as 165±2.8 µM (p<0.001) (Fig. 5d). During early corticogenesis, bursts of action potentials cause spreading of giant waves of calcium influxes through the developing cortex, which is described as giant depolarizing potentials (GDPs), depending both on excitatory glutamate and gammaaminobutyric acid (GABA) inputs. Glutamate-dependent GDP-like events were reported in neuronal organoids. Reduced GDPs in >40 day organoids with GABA polarity switch were regarded as indicators of progressive neuronal network maturation 78 . On the other hand, the glutamate level that secreted from microglia was measured around 20 µM in healthy in vitro neuronal cultures 79,80 . Given that, the change in the uptake and consequently release rate of extracellular glutamate in 60 day RCCS and µ-platform organoids and growth in organoid sizes might be associated with more mature states.
Besides the postsynaptic PSD95 and presynaptic vGLUT1 staining, we newly performed immunofluorescence staining with a presynaptic marker GABA-A to indicate GABAergic interneurons in organoids, and obtained high-quality images under the confocal microscope, which are depicted in Fig. 5a. Additionally, western blot analysis was performed to support result, which can be seen in Fig. 5c. c) In the Discussion section there is a series of overstatements: i. "(1) enriched cellular diversity (abundant neural/glial/ endothelial cell population)" > deeper characterization with higher quality images and more markers should be done to conclude this; Thanks for your comments. Besides rephrasing the sentences in the discussion section, more in-depth characterization was provided in the revised text with high-quality images and FACS, IF, WB and qRT-PCR analyses, as mentioned above.
ii. "(2) structural brain morphogenesis (complex cortical layers, radia-glial organization, preplate splitting)" > no clear demonstration of brain morphogenesis; indeed, the lack of complex structure in brain organoids is one of the limitations of the field. Fig. 4b. Additionally, high-quality confocal images, neural rosette tube-like structures indicated by white arrows and CP layer borders indicated by yellow dashed lines were added to the text.

A clear demonstration was provided with schematic illustration of cellular organization of matured cerebral organoids in
iii. "further functional neuronal identities (glutamatergic and hippocampal neurons) and synaptogenesis (postsynaptic interaction) during whole human brain development" > further analysis using more sophisticated technologies would be necessary to validate this claim.
For further analysis, glutamate measurements, additional IF and WB analysis for GABA-A marker were carried out to validate functional maturation of organoids.
iv. "GFAP+/S100B+ astrocytes and NEUN+/MAP2+ mature neurons were abundant on day 60 in RCCS organoids richest in cell diversity, both RCCS and µ-platform organoids expressed CD11b+/IBA1+ microglia and MBP+/OLIG2+ oligodendrocytes at high levels as of day 60." > further characterization with more markers and higher quality images are necessary to support this claim.
In addition to IF, WB and qRT-PCR analysis, we performed further characterization of cells with FACS analysis. New results have been added to the relevant sections in the revised text.
v. "RCCS organoids with enormous cell architectures, exhibited excellent cortical plate and SVZ architecture at day 120, where TUJ+ cells located at the basal side of the ventricle-like structures with TBR1+ cells located in the deep layer of cortical-like plates as well as well-defined progenitor zone organization, neural identity and further neuronal differentiation with NESTIN+/TBR2+/PAX6+ and SOX1+/SOX2+ cells." > it is not clear what the authors are referring to.
The sentences are rephrased in the discussion section, clear demonstration was provided with schematic illustration of cellular organization of matured cerebral organoids in Fig. 4b, as mentioned above.
vi. "On the other hand, N-CAD+ apical epithelial and TTR+ choroid plexus epithelial cells were populated in RCCS organoids as of day 60, while both FOXG1+ forebrain, KROX20+ hindbrain and PROX1+ hippocampus related to midbrain and hindbrain identities were observed in µ-platform organoids at day 120." > further characterization with more markers and higher quality images are necessary to support this claim.
Thank you, we performed further characterization with higher quality revised images and more markers (β -catenin) and new IF, WB and qRT-PCR analysis (for FOXG1). Furthermore, new β -catenin marker results have been added to the revised text as follows; Vascularization in cerebral organoids was reported to up-regulate the Wnt/β-catenin signaling and increase Ki-67 cell proliferation marker. Endothelial-like cells in vascularized organoids signal to neural stem cells, regulate their self-renewal and differentiation into neurons during CNS development 87 . Additionally, the adherens junctions control vRG cell's self-renewal, proliferation, differentiation and survival via active Wnt/β-catenin/N-cadherin signaling 88 . We also found that both protein and RNA expression levels of β-catenin were more elevated in microgravity driven RCCS organoids and low shear stress induced µ-platform organoids on day 120 (Fig 6a,b, 1. The authors raise a valid point that hemodynamic forces are underexplored in the brain organoid field, despite the known biological relevance of mechanosensitive signals associated with cerebrospinal fluid flow during development. However, the authors do not explore this aspect in their data or discussion. It would be interesting to tie their data back to what is known about this in the neurodevelopmental field.
Within the reviewer's suggestion, we have detailed the results and discussion parts to be more explanatory. Minor concerns 1 and 2 are considered together and explained below 2. The authors hypothesize that microgravity and gravity-driven laminar flows enhance maturation of cerebral organoids recapitulating features of human embryonic cortical development. It would be interesting to discuss the findings that support this hypothesis.
Recently, the transcriptomic analyses of cerebral organoids showed that mechanotransductionassociated genes including integrins, β-catenin, Wnt, and Delta-like pathway genes change under physical stress conditions 98 . Thus, organoids exposed to varying magnitudes of hydrodynamic forces are expected to display differences in structural and functional features during development. As such, cell proliferation of hPSCs has been shown to increase under simulated microgravity culture conditions with significantly higher expression levels of Ki-67 compared to 1g culture condition, leading to enhanced self-renewal, as revealed by the increased protein levels of the core set of pluripotent transcription factors 99 . In another study, human embryonic stem cell (hESC)-derived forebrain-specific neural-cortical organoids were shown to have the highest expression of Ki-67 when cultured under RCCS conditions after day 14 of the generation process compared to organoids cultured in static and other days of the generation in RCCS 100 . These results are in agreement with our study and support the increased expression levels of Ki-67 in RCCS organoids. Also, the use of RCCS under microgravity conditions in embryonic body formation from ESCs has been shown to promote more homogeneous EB formation and endoderm differentiation by modulating the Wnt/β-catenin pathway 101 . On the other hand, the most severe subtype of spina bifida, which is one of the neural tube defects that starts in the 4th week of pregnancy, occurs when the spinal cord is exposed to shear stress from amniotic fluid 102,103 . This negative effect of shear stress on brain and spinal cord formation during embryonic development highlights the importance of shear stress during organoid maturation.
3. "In terms of tissue structure, while the most dispersed organoid structure was observed in static culture and the most uniform distribution was observed in the shaker, the well-defined multi-patterned organoid morphologies with self-organization and desired cellular architectures in the inner-outer regions6,13,28 were observed in the RCCS microgravity bioreactor, followed by µ-platform and spinner systems (Fig.  2b,c,d,e,f)." It is not clear which parameters the authors used to say the latter. How was this quantified?
More clear information about H&E staining added to text as follows and white arrows are added to Fig.2c,d,e,

f,g. to allow better understanding (images with small magnification have been removed to avoid complexity)
In terms of tissue structure, while the most dispersed organoid structure was observed in static culture and the most uniform cell distribution (unorganized) was observed in the shaker, the welldefined multi-patterned organoid morphologies with self-organization and desired cellular architectures in the inner-outer regions6,12,28 were more observed in the RCCS microgravity bioreactor (sequential organized cell nucleus and neural rosette-like structure indicated by white arrows), followed by µ-platform and spinner systems (Fig. 2c,d,e,f,g). Fig.2b and organoid size graphs in Fig.2c,d,e,f,g.

What does "neurons at different polarized states" mean?
This statement is detailed as follows;

During human embryonic brain development, neurons and glial cells are derived from neural stem/progenitor cells that generate specific brain regions and cortical layers. The derivation is achieved by an asymmetric spatial organization of cells and different components, explained as cell polarity. In the polarization process, the ventricular zone (VZ) formed by neuroepithelial cells is followed by the subventricular zone (SVZ), where apical progenitors form basal progenitors, and then cortical plate (CP) by apical and basal progenitors forming neurons 50 . The degree of efficient differentiation is determined by the expression of neurons at different polarized states and progenitor specific markers.
5. Authors mentioned the presence of "cortical plate" in their organoids, but this is not clear from the images provided. Fig.4a, a new staining was carried out and high-quality images demonstrating a high proportion of FOXG1+ and PAX6+ cells were added to assess the quality of neuronal cell differentiation in Fig.6a. Also, white arrows added to indicate TBR1+/PAX6+/SOX2+/FOXG1+ and DAPI+ neural rosette and neural tube-like structures in Fig. 4a, 6a,b and Supp. Fig. 2 Fig.4b. Then, yellow dashed lines were added to indicate cortical plate layer borders in Fig.4a,5a, between the early-born neurons expressed CTIP2 in the deep-layer and the late-born neurons expressed SATB2 in the superficial upper-layer.

. Next, a schematic illustration and new literature information was added to text to show cellular organization of matured cerebral organoids as
6. "Paired box protein 6 (PAX6) is a transcription factor expressed in neural stem cells and ventral forebrain radial glial progenitor cells. Although expressed in all brain regions, the highest expression level is reported at the cerebellum, which is involved in glial cell differentiation55." This sentence is confusing: it is unclear the relationship between glial cell differentiation and the cerebellum.

Paired box protein 6 (PAX6) is a transcription factor expressed in neural stem cells along with ventral forebrain radial glial progenitor cells and has an important role in the differentiation of radial glial cells located in different regions of the CNS, such as the cerebral cortex, cerebellum, forebrain and hindbrain 59 .
7. "CTIP2, SATB2, and TBR1 were upregulated at protein and RNA levels which indicated cortical neuronal differentiation in both RCCS and µ-platform organoids mainly on day 120 (Fig. 4, Supp. Fig. 3), supporting the spatial distribution and functional organization of cortical neurons61." It has been demonstrated in 3D brain organoid cultures that generation of the different cortical neuron subtypes is not necessarily accompanied by structural/functional organization in the stereotypical layers of the human cerebral cortex (PMID: 24277810). Therefore, this sentence should be revised.

The sentence is revised as follows:
CTIP2, SATB2, and TBR1 were upregulated at protein and RNA expression levels in both RCCS and µ-platform organoids mainly on day 120 (Fig. 4, Supp. Fig. 3, Supp. Table 5), showing an insideout pattern of CP (yellow dashed lines indicate CP layer borders). Early-born neurons expressed CTIP2 and TBR1 in the deep-layer, whereas late-born neurons expressed SATB2 in the superficial upper-layer with the obvious spatial zone separation 65,66 8. In the methods, the authors mentioned that some modifications were done to the organoid protocol, but they do not mention what was done.

The sentence is revised as follows to avoid any confusion;
The cerebral organoid generation steps were based on the detailed method of Lancaster and Knoblich 116 with some minor modifications regarding the durations in consecutive steps starting from maturation, in which dynamic systems and flow rates are different from the applied method.

Reviewer #2 (Remarks to the Author):
Saglam-Metiner et al. reported that RCCS microgravity bioreactor and μ-platform presented improvement of the harvestability, scalability, reproducibility, and survival of cerebral organoids, as well as functional maturation. It is assumed that comparing these various 3D culture methods with each other required a lot of effort, and the results should provide important information to the scientific research field. However, their findings are just observations, and quantitative data are lacking. Statistical analysis of quantitative data is a basic requirement for scientific consideration. Furthermore, there are no data to allow the evaluation of physiological function, although the authors mentioned that their organoids have achieved functional maturity. There remain many issues to be resolved before reaching the conclusion the authors proposed.

Thanks for the comments and suggestions, which helped to increase the quality of the manuscript. Please find our point-by-point responses to each of the comments raised.
Major points: 1. The authors should add the quantitative data to explain the improvement of harvestability, scalability, reproducibility, and survival of cerebral organoids to compare the respective culture conditions, and they should present the results of statistical analysis.
Thank you for the comment. We added a graph and statistical analysis to highlight the harvestability results in Fig. 2b. The sentences are revised in the text as follows; On the 15th day of culture, generated organoids (derived from two different passage numbers of iPSCs at two different times) were transferred to RCCS, µ-platform, spinner and shaker, along with static culture as the control group (considered as day 0 of maturation), in order to determine the best dynamic system for physical and functional maturation of cerebral organoids, which were cultured for 120 days. Organoid size distribution variability is known to be a limitation of traditional human brain organoid protocols, as well as cellular composition and organization. Therefore, organoid reproducibility and batch-to-batch variability was firstly examined in terms of harvestability, macroscopic-microscopic observations and organoid size distribution (Fig.  2b,c,d,e,f,g). As such, cellular composition and cellular organization were assessed with cell sorting, immunostaining, western blot, qRT-PCR, TUNEL and glutamate secretion analyses for organoids sampled on various days (30, 60 and 120th days).
We used the ImageJ programme to quantify apoptotic zone vs nucleus, data were then calculated with GraphPad Prism 8.3.0. The quantitative results of apoptotic zone % in Fig. 6d with statistical analysis were added to the revised text.
With respect to the scalability, the sentences in the discussion section were corrected as follows to eliminate the ambiguity; Although scale-up was not planned for this study, we presume that about 1000 matured organoids can be harvested in one batch, if we scale up to 500 ml RCCS STLVs, as such the µ-platform can be multiplied by connecting in a parallel or serial manner for scale-up purposes.
2. Although the authors described organoid-maturation, they only presented immunostaining and western blot data to show protein expression related to synapses such as PSD95. The authors should present the data of physiological analysis to show that their organoids have acquired functional maturation as neurons.
Thanks for the suggestion. Besides, postsynaptic PSD95 and presynaptic vGLUT1 staining, we additionally performed immunofluorescence staining with a presynaptic marker GABA-A to indicate GABAergic interneurons in organoids, and obtained high-quality images under the confocal microscope. Fig. 5a. and details about GABA-A is provided in the results section as follows. Moreover, western blot analysis was performed to support results in Fig. 5c.

During early corticogenesis, bursts of action potentials cause spreading of giant waves of calcium influxes through the developing cortex, which is described as giant depolarizing potentials (GDPs), depending both on excitatory glutamate and gamma-aminobutyric acid (GABA) inputs.
Glutamate-dependent GDP-like events were reported in neuronal organoids. Reduced GDPs in >40 day organoids with GABA polarity switch were regarded as indicators of progressive neuronal network maturation 78 . On the other hand, the glutamate level that secreted from microglia was measured around 20 µM in healthy in vitro neuronal cultures 79,80 . Given that, the change in the uptake and consequently release rate of extracellular glutamate in 60 day RCCS and µ-platform organoids and growth in organoid sizes might be associated with more mature states. One of the distinguishing features of neuronal circuit and maturation is the switch of excitatory to inhibitory GABAergic neurotransmission. GABA-A, a presynaptic GABAergic interneuron receptor that modulates neurotransmitter release in both peripheral and central synapses 7,78,[81][82][83] , was more expressed in both RCCS and µ-platform organoids with CTIP2+ deep-layer of cortical neurons as of day 60 (Fig. 5a,b,c). Thus, the neuronal network complexity was validated in RCCS and µ-platform organoids with the presence of both presynaptic glutamatergic VGLUT1+ and GABAergic GABA-A+ neurons which are critically involved in neuronal network oscillations, as well as postsynaptic PSD95+ neurons, GFAP+/S100B+ astrocytes and MBP+/OLIG2+ oligodendrocytes.
Additionally, we performed glutamate analysis to examine molecular and functional maturation of RCCS and platform organoids as depicted in Fig.5d and details are given in the methods and results section, as mentioned before.
3. Although the authors mentioned that a layered structure of brain has formed, the figures showed no such structure but only a scattering of cells expressing markers for the cortical layers. The authors should correct the description.
Within the reviewer's valuable suggestion, a new staining of FOXG1+ and PAX6+ cells were added to assess the quality of neuronal differentiation in Fig.6a and white arrows added to indicate TBR1+/PAX6+/SOX2+/FOXG1+ and DAPI+ neural rosette and neural tube-like structures in Fig. 4a,6a,b and Supp. Fig. 2. Also, a schematic illustration and new literature information was added to text to show cellular organization of matured cerebral organoids with respect to MZ, CP, oSVZ, SVZ, VZ zones as depicted in Fig.4b. Yellow dashed lines were added to indicate cortical plate layer borders in Fig.4a,5a, between the early-born neurons expressed CTIP2 in the deep-layer and the late-born neurons expressed SATB2 in the superficial upper-layer.
4. The authors should provide the details of the equipment, and especially the μ-platform that was reported as being newly constructed in this paper.
Thanks, "Supp. Fig. 1" has been revised by adding all system illustrations and detailed with the size/volume information used in the simulation.
5. The authors should compare the cell-type characteristics and maturation status in single-cell resolution methods like single-cell RNA sequence or detailed IFC investigation because organoid contains various types of brain cells. Quantification of these data will be helpful for understanding the beneficial points of the presented platform.
We appreciate the reviewer's insightful suggestion. Besides detailed IF, WB and qRT-PCR analysis, the manuscript has been much improved within the newly performed quantitative FACS analysis to examine important types of brain cells such as CD45+/CD11b+ microglia and GFAP+ astrocyte in RCCS organoids, which is presented in Fig. 3d 6. The authors attempted to characterize different culture platforms by hydrodynamic analysis. This analysis clarifies the characteristics of how the medium moves and can be considered to be useful data. However, what this analysis reveals is limited to phenomena outside of organoids, such as the distribution of shear stress. This analysis unfortunately does not help us to understand what physical effects occur inside individual organoids and, as a result, affect differentiation and maturation. Care should be taken so as not to overstate this point, and to improve the description to avoid misunderstandings.
Thanks for pointing this out, we agree that the flow simulation just characterizes the hydrodynamics (velocity, shear stress, flow distribution, etc.) acting on the organoids. However, the purpose of this work was not related to clarifying the underlying mechanisms of mechanotransduction signaling. Herein, the effects of different external fluid dynamics on maturation in cerebral organoids were examined with gene and protein expression levels. Furthermore, we believe that this work will shed light on future studies that will show which pathways responsible for mechanotransduction are activated by transcriptomic analyzes. With your comment, we avoided overstatements in the text and added more in-depth discussion as follows; Physical forces, such as shear stress, gravity and cyclic stretch affect mechanosensitive pathways or change the expression levels 22 . Recently, the transcriptomic analyses of cerebral organoids showed that mechanotransduction-associated genes including integrins, β-catenin, Wnt, and Delta-like pathway genes change under physical stress conditions 98 . Thus, organoids exposed to varying magnitudes of hydrodynamic forces are expected to display differences in structural and functional features during development. As such, cell proliferation of hPSCs has been shown to increase under simulated microgravity culture conditions with significantly higher expression levels of Ki-67 compared to 1g culture condition, leading to enhanced self-renewal, as revealed by the increased protein levels of the core set of pluripotent transcription factors 99 . In another study, human embryonic stem cell (hESC)-derived forebrain-specific neural-cortical organoids were shown to have the highest expression of Ki-67 when cultured under RCCS conditions after day 14 of the generation process compared to organoids cultured in static and other days of the generation in RCCS31. These results are in agreement with our study and support the increased expression levels of Ki-67 in RCCS organoids. Also, the use of RCCS under microgravity conditions in embryonic body formation from ESCs has been shown to promote more homogeneous EB formation and endoderm differentiation by modulating the Wnt/β-catenin pathway 100 . On the other hand, the most severe subtype of spina bifida, which is one of the neural tube defects that starts in the 4th week of pregnancy, occurs when the spinal cord is exposed to shear stress from amniotic fluid 101,102 . This negative effect of shear stress on brain and spinal cord formation during embryonic development highlights the importance of shear stress during organoid maturation.

Minor points
1. Signal/background ratios of images are not enough. Revise the setting when acquiring images Thanks for the comment. The quality and resolution of all figures has been increased and revised in the illustration program. If required, all figures can be uploaded in tiff format, separately from the manuscript.
2. The shape and marginal border of organoids are irregular, and these organoids appeared to be dying in Figures 3-6. The authors can investigate and describe what happens, e.g. cellular migration, cell death,

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